CN202676705U - Combined insect-resistant BT protein gold mark fast detection reagent kit - Google Patents
Combined insect-resistant BT protein gold mark fast detection reagent kit Download PDFInfo
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- CN202676705U CN202676705U CN 201220069968 CN201220069968U CN202676705U CN 202676705 U CN202676705 U CN 202676705U CN 201220069968 CN201220069968 CN 201220069968 CN 201220069968 U CN201220069968 U CN 201220069968U CN 202676705 U CN202676705 U CN 202676705U
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Abstract
The utility model relates to a combined insect-resistant BT protein gold mark fast detection reagent kit. The reagent kit comprises a bottom plate and an upper cover, wherein 4 to 8 testing bands are arranged in the bottom plate, and 4 to 8 observing windows and 4 to 8 sample-adding holes are arranged in the upper cover. The reagent kit can detect various transgenic components simultaneously at a time without need of other additional devices and equipment. The reagent kit is simple to operate, and visual, accurate and fast in detection result. As long as extract liquid of detected objects is dropped in the 4 to 8 sample-adding holes of the reagent kit, accurate detection results can be visually observed from the observing windows with naked eyes in 5 to 10 minutes.
Description
Technical field:
The utility model relates to the pest-resistant BT albumen of combined type gold labeled quick detection reagent box.
Background technology:
In the developments of genetically modified plants or transgenic product is being screened or during safety evaluatio, often needing the allogenic gene that changes over to is carried out qualitative or semiquantitative determination.Sometimes also need multiple allogenic gene is detected simultaneously.
Bacillus thuringiensis (BT) has widely insecticidal activity, has found at present more than 800 kind of insecticidal crystal protein.Along with the development of DNA recombinant technique, the Bt insecticidal crystalline gene is imported in other microorganisms or the plant, structure can produce the engineered strain of insecticidal proteins or main direction (Barton, the et al.1987 that insect-resistant transgenic plants becomes research; Fischhoff et al.1987; Vaeck, et al.1987; Perlak, et al.1990).Approximately nearly 20 kinds of Bt insecticidal crystalline genes have changed different plants over to.Turned in the first batch pest-resistant tobacco and tomato appearance (Barton, the et al.1987 of Bt cry gene in 1987; Fischhoff, et al.1987; Vaeck, et al.1987).Transgenic Bt cotton, corn and potato in 1996 are in U.S.'s commercialization plantation (Peferoen 1997) that goes through, and this indicates that the application of bt insecticidal crystal protein enters the new industrialization stage.But the amino acid sequence of these BT insecticidal crystal proteins is widely different, and for example the homology of Cry1AC and Cry2AB is 45%; The homology of Cry1Aa and Cry3A only has 25-30% etc. (Li et al., 1991; Grochulski et al., 1995).
Existing pick-up unit, exempt from, put the device of methods such as exempting from such as enzyme, be subjected to the restriction of the factors such as instrument (microplate reader, put and exempt from scintiloscope, hydro-extractor etc.), place, and detection time is long, and (the inspection-free survey time of enzyme needs 20hr, put and need to exempt from 30min～1hr), testing cost is higher, is difficult to apply.Though the method for ELISA test strip can partly overcome above deficiency (such as U.S. EnviroLogix Incorporated in the past, Strategic Diagnostics, Inc. mark the test strips that Bioisystech Co., Ltd produces with Beijing wound gold difficult to understand), but every kind of test strips can only detect foreign gene single in the genetically modified crops, easily produces error result in the practical operation.For example: if the tester only has Bt Cry1Ac test strip, and do not change BT cry1Ac gene in the detected object over to, but change the genes such as Bt cry 2ab over to, and draw so possibly the error result that there is not foreign gene in detected object, cause unnecessary loss.
Therefore, in the practice in the urgent need to a kind of different B t crystalline protein that can detect simultaneously in the genetically modified plants is arranged, and easy and simple to handle, result accurately and reliably, device fast.
The utility model content:
The purpose of this utility model is to set up a kind of easy and simple to handle, result accurately and reliably, fast, can detect simultaneously the combined reagent box of multiple trans Bt gene composition.
The technical solution of the utility model is such:
The pest-resistant BT albumen of a kind of combined type gold labeled quick detection reagent box comprises base plate, loam cake, and base plate cover forms kit after entering loam cake; It is characterized in that being placed with on the base plate calibration tape; Loam cake is provided with observation window and well;
The upper surface of described base plate is provided with 4～8 grooves that can embed a calibration tape;
Described calibration tape quantity is 4～8; The quantity of described observation window and well is respectively 4～8.
Described base plate or loam cake can have draw-in groove, to guarantee base plate and loam cake fastening.
Described observation window is rectangle preferably.
The shape of kit is flat fan-like pattern box.
In an example of the present utility model, can detect simultaneously 6 kinds of trans Bt gene compositions, in base plate, can put 6 different calibration tapes, and loam cake arranges observation window and the well of respective numbers.
6 calibration tapes in the described base plate, article one, for detection of BT-Cry 1Ac albumen, second is for detection of BT-Cry1Ah albumen, article three, for detection of BT-Cry 2AB albumen, article four, for detection of BT-Cry 9C albumen, article five, for detection of BT-Cry 3A albumen, the 6th for detection of BT-Cry 1Aa albumen.Described calibration tape is a kind of test material with detection system body membrane reaction system function, is made by multilayer materials such as macromolecular fibre film, plastics.The calibration tape of six kinds of albumen all can be divided into Quality Control district and detection zone, and the Quality Control district can be coated with sheep anti-mouse igg or rabbit anti-mouse igg polyclonal antibody; Monoclonal antibody or the polyclonal antibody of the coated BT-Cry 1Ac in the sample detection district of BT-Cry 1Ac albumen calibration tape; Monoclonal antibody or the polyclonal antibody of the coated BT-Cry 1Ah in the sample detection district of BT-Cry1Ah albumen calibration tape; Monoclonal antibody or the polyclonal antibody of the coated BT-Cry 2AB in the sample detection district of BT-Cry 2AB albumen calibration tape; Monoclonal antibody or the polyclonal antibody of the coated BT-Cry 9C in the sample detection district of BT-Cry 9C albumen calibration tape; Monoclonal antibody or the polyclonal antibody of the coated BT-Cry 3A in the sample detection district of BT-Cry 3A albumen calibration tape; Monoclonal antibody or the polyclonal antibody of the coated BT-Cry 1Aa in the sample detection district of BT-Cry 1Aa albumen calibration tape.
When different foreign genes change plant over to, and in plant, express, to produce different destination proteins (BT-Cry 1Ac, BT-Cry1Ah, BT-Cry 2AB, BT-Cry 9C, BT-Cry 3A and BT-Cry 1Aa), therefore determine by whether containing above-mentioned six kinds of albumen in the test sample whether this gene exists.If foreign gene changes plant over to, producer is reticent, produces without destination protein, and (desinsection etc.) can not accomplish the end in view.
The purpose of this utility model is achieved in that after base plate cover enters loam cake, each respectively corresponding calibration tape of each observation window and well, can carry out application of sample and observe testing result by the well on the lid and observation window, each observation window can check respectively a kind of gene.
Each observation window is divided into test section (T) and Quality Control district (C) two parts, can indicate covering with letter C, T;
During detection, the same extract of censorship article is dripped respectively in six wells of kit, behind the 5-10min, with the naked eye the observation window by loam cake can observe directly accurately testing result.
For each observation window, judge with the following methods testing result:
1, negative (-):
Reaction condition: an aubergine band appears in observation window Quality Control district (C).
Testing result: do not contain gene to be checked or its content in the sample to be checked at it below threshold value.
2, positive (+):
Reaction condition: an aubergine band appears in observation window Quality Control district (C), the aubergine band occurs simultaneously in test section (T).
Testing result: gene content to be checked is more than threshold value.
3, invalid:
Reaction condition: the aubergine band does not appear in Quality Control district (C).
Testing result: show rotten damage of incorrect operating process or kit.
The advantage of kit described in the utility model is:
Economical and efficient: a detection box can detect 6 kinds of different transgenosis compositions simultaneously, greatly reduces use cost;
Easy to be quick: 6 kinds of different transgenosis composition single stage method operations, detect simultaneously, do not need other any auxiliary instrumentation equipment.Directly analyte sample fluid is dripped in well, 5-10min can go out the result;
Testing result is directly perceived: not by any instrument, with the naked eye can be observed the result;
Be widely used: blade, seed and the converted products that can detect the multiple kinds of crops such as corn, cotton, tobacco, rape, soybean, paddy rice.
Because can carrying out BT-Cry 1Ac, BT-Cry 1Ah, BT-Cry 2AB, BT-Cry 9C, BT-Cry 3A and BT-Cry 1Aa to plant sample simultaneously, the pest-resistant BT albumen of the combined type gold labeled quick detection reagent box that the utility model provides analyzes, detect quick, special, responsive, convenient, be specially adapted to the development of transgenic product and the rapid screening of transgenic product, can be used for frontier defense, customs's plnat monitoring, food safety detection and seed and manage sales department scene, rapid screening testing sample.
Below in conjunction with drawings and Examples, further illustrate technology contents of the present utility model.
Description of drawings:
Fig. 1 and Fig. 2 are the structural representations of the pest-resistant BT albumen of the utility model combined type gold labeled quick detection reagent box, wherein:
Fig. 1 is the vertical view of base plate, and base plate has six calibration tapes; Among the figure, the 3rd, calibration tape.
Fig. 2 is the vertical view of kit loam cake, on be stamped 6 observation windows; Among the figure, the 1st, well, the 2nd, observation window.
Embodiment:
The preparation of the calibration tape in the pest-resistant BT albumen of the utility model combined type gold labeled quick detection reagent box brings with the test for preparing BT-Cry 1Ah and illustrates:
1.BT-Cry the preparation of 1Ah odd contradictive hydroperitoneum
Mouse peritoneal injecting fluid paraffin 0.5mL.After 1 week, BT-Cry 1Ah monoclonal antibody hybridoma is injected in above BALB/C mice abdominal cavity of anticipating, every injected in mice hybridoma 1～3 * 10
6Gather in the crops ascites after 10 days, above process is all finished under aseptic condition.
2.BT-Cry the purifying of 1Ah monoclonal antibody
1) with DEAE ion-exchange chromatography and Sephacryl S 300 molecular sieve monoclonal antibody is carried out purifying; The SDS-PAGE disk electrophoresis detects the purity of purified monoclonal antibody; It is active that the ELISA method is measured monoclonal antibody;
2) purge process: get the centrifugal 15min → supernatant of mouse odd contradictive hydroperitoneum → 3,000 * g and add 50% (NH
4)
2SO
4Precipitation, spend the night → that the centrifugal 20min of 3,000 * g → precipitation is dissolved in 0.01M phosphate buffer (pH 7.2) → S-300 gel column → DEAE Blue chromatographic column → 0-800mM NaCl gradient elution → ultrafiltration is centrifugal, concentrates monoclonal antibody;
3) monoclonal antibody purity testing: conventional SDS-PAGE electrophoresis, the purity of the monoclonal antibody that said method is purified are all more than 95%;
4) determination of protein concentration: ultraviolet spectrophotometer is measured the OD value at 279nm place, calculates as follows protein content: OD
279/1.4=mg/mL (purified monoclonal antibody).Be about 1mg/mL with the monoclonal antibody concentration adjustment;
5) monoclonal antibody determination of activity: select the antigen coated elisa plate of BT-Cry 1Ah (1 μ g/ hole), and sheep anti-mouse igg-HRP compound, measure each monoclonal antibody tire (greater than 1: 5000).
3. goat-anti DOA
TThe high-titer sero-fast preparation of immunity and purifying
1) above-mentioned monoclonal antibody+Freund's complete adjuvant that purifying is good → immune goat → booster immunization 2 times, every minor tick 1 month → strengthen was for the second time got blood → centrifuging and taking serum → (NH in rear about 10 days
4)
2SO
4Precipitation → DEAE ion-exchange chromatography purifying;
2) titration: the ELISA sandwich method, the antibody titer dilutability should be greater than 1: 256;
3) determination of protein concentration: ultraviolet spectrometry is measured OD
279, calculate protein concentration.
4. collaurum and golden labeling antibody preparation
1) preparation of collaurum: the collaurum for preparing 40-60nm with the citrate reducing process.With 0.01%HauCl
4Be heated to and boil, add a certain amount of 1% trisodium citrate (Na
3C
6H
5O
72H
2O), continue heating and boil 5min, treat the colloidal gold solution color by blue → purple → red, stable after, cooling gets final product.Colloidal gold solution should be limpid transparent, such as the need long preservation, can add 0.02%NaN
3
2) collaurum liquid 500mL is transferred pH 84 with 0.1M NaOH, slowly add monoclonal antibody 2mL under the magnetic agitation, stir 20min.The centrifugal 30min of 5,500 * g removes unconjugated protein in the supernatant.Centrifugal rear absorption colloidal gold labeled monoclonal antibody precipitation, precipitation is dissolved in 10mL and preserves in the liquid, with 0.45 μ m filtering with microporous membrane.Sampling is examined and determine, and all the other put 4 ℃ of preservations.
5. the preparation of film reaction system M1
1) with the sheep anti-mouse igg of BT-Cry 1Ah antibody and purifying with the dilution of 0.1M phosphate buffer, final concentration is about 0.2-2mg/mL.
2) sheep anti-mouse igg of purifying is dissolved in the 0.1M phosphate buffer, and concentration is 2.0mg/mL;
3) nitrocellulose filter size: 10cm * 27cm, every film can spray the detection of long 25cm and control each 4 on band;
4) above two kinds of solution are added respectively in 2 shower nozzle storage bottle of Biodot XYZ3000 flush coater;
5) spray speed being set is 2 μ L/cm, and the speed of dividing a word with a hyphen at the end of a line of NC film is 50mm/s.At the coated reaction detection line of NC film, with the coated reaction of 1.0mg/mL rabbit anti-mouse igg control line, the distance of control line and detection line is 4-4.5mm with monoclonal antibody Cry 1AhAD2 (2mg/mL);
6) finish coated after, put 37 ℃ of drying box 24h, process 0.5h with the sealing of BB confining liquid, take out with the WB washing lotion and wash 1 time;
7) 37 ℃ of drying box drying for standby;
8) nitrocellulose filter got ready of cutting: 1.8cm * 27cm/ bar, put into the thin aluminum bag hermetically drying and preserve.Put room temperature preservation for subsequent use.
6. the preparation of film reaction system M2a, M2b and M3
The water-absorption fiber film is soaked in respectively in M2a, M2b, the M3 solution, and after soaking into, taking-up is dried, in the polybag of packing into, and room temperature preservation.
1) M2a preparation: the glass fibre that makes is cut 27cm * 1.2cm/ bar;
2) M2b preparation: the glass fibre that makes is cut 27cm * 1.8cm/ bar;
3) M3 preparation: the glass fibre that makes is cut 27cm * 1.2cm/ bar.
7. the preparation of film reaction system M4
1) get collaurum-monoclonal antibody compound and add dilution, mixing is made into working concentration;
2) solution is added in the Airjet shower nozzle storage bottle of flush coater Biodot;
3) set pressure is 15PSI, and the translational speed of glass fibre membrane is 50mm/s;
4) spray specification is 0.5-1.5cm * 25cm/ bar, and every sprays 2 times;
5) in 37 ℃ of oven dry 12h, put into thin aluminum bag, add drying agent, heat sealing, room temperature preservation.
8. reaction body assembling
1) pastes 6 double sticky tape M7 at the M6 plastic base;
2) paste reaction film M1 (18mm) in the middle of the M7, from the about 22mm of M6 upper limb;
3) paste M5 (22mm) at M7, align with the M6 upper limb and hand over 1mm with the M1 upper limb;
4) paste M2a (12mm) at M7, join with the M1 lower edge;
5) paste M4 (10mm) at M2, push down M1 lower edge 0.5mm;
6) paste M3 (12mm) at M7, upper limb is pushed down M4 approximately 2/3;
7) paste M2b (18mm) at M7, upper limb is pushed down M4 approximately 2/3, and lower edge aligns with the lower edge of M7;
8) paste transparency protected adhesive tape M8 at M4 and M7, upper limb is pushed down M4 fully, and pushes down approximately 1.5mm of M1;
9) paste colour-coded adhesive tape M9 at M5, hand over 1mm with the M1 upper limb, the other end climbs over the M6 upper limb, is affixed on the M6 quilt cover;
10) the reaction body and function full-automatic strip cutter with assembled formation is cut into the 3.5mm specification.
The assembling of embodiment 2 kits
Six kinds of calibration tapes of detection BT-Cry 1Ac, the BT-Cry 1Ah, BT-Cry 2AB, BT-Cry 9C, BT-Cry 3A and the BT-Cry 1Aa albumen that prepare are embedded respectively in six grooves of base plate, then with loam cake and base plate fastening, kit.
Behind kit, dropper and operation instructions dress packaging plastic bags, get final product.
1. detect sample process and preparation
Vegetable seeds, leaf, seedling equal samples need through grinding before detection, and with distilled water extracting and dilution.For obtaining the optimum detection effect, variant sample should after finishing grinding and dilution, with the sample mixing, leave standstill according to the dilution proportion in the following table, gets supernatant as test sample.
2. detect and the result
To detect box and keep flat, and with joining plastic suction pipe absorption sample liquid, drip 4-5 and drip in the well 1 that detects box, absorbent material makes sample to be checked slowly mobile, and the film reaction system is activated.No matter whether testing gene is present in the plant sample, and an aubergine band all can appear at (C) in the Quality Control district.The aubergine band that (C) manifests in the Quality Control district is to determine whether enough plant sample liquid, and whether chromatography process normal standard, simultaneously also as the inner quality standard of reagent.
3. the result judges:
Whether each observation window detects a kind of gene to be checked and exists.Below to detect BT-Cry 1Ah as the example explanation:
1) as not containing particular B T-Cry 1Ah albumen in the sample, or its concentration is lower than detection sensitivity, colloidal gold antibody can not be fixed on the monoclonal antibody immunity in the film detection band in chromatography process, thereby an aubergine detection band can not appear in (T) in the test section, show negative (-) result, an aubergine band namely only in Quality Control district (C), occurs.
2) if when treating that BT-Cry 1Ah protein concentration is higher than its detection threshold in the plant sample, antigen immunity gold with detect with on another monoclonal antibody be combined, another aubergine detection band will also appear in (T) in the test section, show positive (+) result, an aubergine band namely in Quality Control district (C) and detection zone, respectively occurs.Attention: the aubergine band in test section (T) can show the phenomenon of shade.But, within the observing time of regulation, no matter this colour band shade all should be judged to be positive findings.
3) the aubergine band do not occur such as Quality Control district (C), then testing result is invalid, shows rotten damage of incorrect operating process or kit.
Claims (2)
1. the pest-resistant BT albumen of a combined type gold labeled quick detection reagent box comprises base plate, loam cake, and base plate cover forms kit after entering loam cake; It is characterized in that the kit shape is flat fan-like pattern box, and be placed with 4～8 calibration tapes on the base plate; Loam cake is provided with 4～8 observation windows (1) and 4～8 wells (2).
2. the pest-resistant BT albumen of combined type according to claim 1 gold labeled quick detection reagent box is characterized in that the upper surface of described base plate is provided with 4～8 grooves that can embed a calibration tape.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220069968 CN202676705U (en) | 2012-02-28 | 2012-02-28 | Combined insect-resistant BT protein gold mark fast detection reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220069968 CN202676705U (en) | 2012-02-28 | 2012-02-28 | Combined insect-resistant BT protein gold mark fast detection reagent kit |
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| Publication Number | Publication Date |
|---|---|
| CN202676705U true CN202676705U (en) | 2013-01-16 |
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| CN 201220069968 Expired - Lifetime CN202676705U (en) | 2012-02-28 | 2012-02-28 | Combined insect-resistant BT protein gold mark fast detection reagent kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116359498A (en) * | 2023-02-23 | 2023-06-30 | 兰州兽研生物科技有限公司 | Immune chromatography test paper card for joint inspection of foot-and-mouth disease, african swine fever, swine fever and porcine reproductive and respiratory syndrome virus antibodies |
| CN117143831A (en) * | 2023-10-31 | 2023-12-01 | 中国农业科学院生物技术研究所 | Insect-resistant protein hybridoma cell strain, antibody produced by same and application thereof |
| CN117147882A (en) * | 2023-10-31 | 2023-12-01 | 中国农业科学院生物技术研究所 | Kit and method for quantitative detection of insect-resistant protein Cry1Ah enzyme-linked immunity |
| CN117169516A (en) * | 2023-11-01 | 2023-12-05 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof |
-
2012
- 2012-02-28 CN CN 201220069968 patent/CN202676705U/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116359498A (en) * | 2023-02-23 | 2023-06-30 | 兰州兽研生物科技有限公司 | Immune chromatography test paper card for joint inspection of foot-and-mouth disease, african swine fever, swine fever and porcine reproductive and respiratory syndrome virus antibodies |
| CN116359498B (en) * | 2023-02-23 | 2024-06-11 | 兰州兽研生物科技有限公司 | Immune chromatography test paper card for joint inspection of foot-and-mouth disease, african swine fever, swine fever and porcine reproductive and respiratory syndrome virus antibodies |
| CN117143831A (en) * | 2023-10-31 | 2023-12-01 | 中国农业科学院生物技术研究所 | Insect-resistant protein hybridoma cell strain, antibody produced by same and application thereof |
| CN117147882A (en) * | 2023-10-31 | 2023-12-01 | 中国农业科学院生物技术研究所 | Kit and method for quantitative detection of insect-resistant protein Cry1Ah enzyme-linked immunity |
| CN117147882B (en) * | 2023-10-31 | 2024-01-26 | 中国农业科学院生物技术研究所 | Kit and method for quantitative detection of insect-resistant protein Cry1Ah enzyme-linked immunity |
| CN117143831B (en) * | 2023-10-31 | 2024-01-26 | 中国农业科学院生物技术研究所 | Insect-resistant protein hybridoma cell strain, antibody produced by same and application thereof |
| CN117169516A (en) * | 2023-11-01 | 2023-12-05 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof |
| CN117169516B (en) * | 2023-11-01 | 2024-02-23 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry1Ah colloidal gold immunochromatography rapid test strip and detection method thereof |
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| GR01 | Patent grant | ||
| CX01 | Expiry of patent term |
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| CX01 | Expiry of patent term |